The fabrication of cell culture substrates in 2-D with tunable stiffnesses has been an intensively studied field in mechanobiology. Previous studies have revealed that cell motility, the reorganization of cellular cytoskeleton and stem cell differentiation are influenced by substrate stiffness and compliance.
Polymeric substrates which have been used in extracellular substrate stiffness studies include poly(ethylene glycol) (PEG) gels, polydimethylsiloxane (PDMS), polyacrylamide (PA) and hyaluronic acid (HA) based polymer systems. These polymers require the establishment of unique formulations for each gel system, such as tuning the different cross-linker concentrations to vary the stiffness of the gel system. A major disadvantage and limitation of these systems are that the material properties are fixed and not dynamic. As such, spatially dynamic stiffness gradients within hydrogels have been formulated to overcome these limitations. For example, hydrogels with differing stiffnesses on the same substrate have been fabricated by mixing different formulations of PA gels. More advanced fabrication techniques have made use of microfluidics to fabricate PA gel gradients. PDMS substrates with stiffness gradients have also been fabricated from patterning. Although substrate stiffness properties can be dynamically controlled spatially in these cases, the limitation of static temporal stiffness in these systems still exists.
To overcome the limitation in temporal stiffness experienced by existing polymeric systems, ultra-violet (UV) photomodulatable hydrogels have been used, such as PA, PEG and methacrylated HA. However, these systems still face a limitation as the change in stiffness is not reversible spatially or temporally. The stiffness of the substrates can only soften or stiffen with time.
Present methods to fabricate thin-film polymers include the spin coating of PDMS as well as the fabrication of thin PDMS films from silicon wafer molds. Disadvantages to current thin-film fabrication methods are that the direct spin coating or pouring of PDMS into molds would cause the PDMS to stick to the underlying substrate, and the PDMS thin-film is subjected to a large peeling force when the film is removed from the substrate, possibly tearing it. Even if the films are able to be removed, the lateral dimensions of the films are limited to hundreds of microns in length.
In addition, current existing methods for fabricating bilayer polymeric substrates suitable for 2-D cell culture include polyelectrolyte multilayer (PEM) fabrication by depositing polyanions and polycations separately onto a surface and the successive spin coating of polymers on top of one another on a rigid substrate. Such methods do not allow the separation of the individual layers away from the bilayer structure.
Of the known apparatus and systems mentioned above, none can be used to induce different cell growth characteristics (i.e. such as morphology, motility, aggregation, differentiation etc.) in a reversible manner such that the induction of different growth characteristics can be dynamically changed and selected.
There is therefore a need to provide an apparatus or method that at least partially ameliorates one or more of the disadvantages described above.